Kahalalide f formulation

ABSTRACT

New formulations and new uses of kahalalide F are provided.

[0001] The present invention relates to kahalalide F, a peptide isolatedfrom a herbivorous marine species of mollusc, Elysia rufescens.

BACKGROUND OF THE INVENTION

[0002] Kahalilide F is the subject of European Patent 610,078. Thepatent reports activity against in vitro cell cultures of human lungcarcinoma A-49 and human colon carcinoma HT-29.

[0003] More information concerning kahalalide F is to be found, forexample, in:

[0004] The absolute stereochemistry of kahalalide F. Goetz, Gilles;Yoshida, Wesley Y.; Scheuer, Paul J. Dep. Chemistry, Univ. Hawaii,Honolulu, Hi., USA Tetrahedron (1999), 55(25), 7739-7746.

[0005]  [Erratum to document cited in CA131:157974]. Tetrahedron (1999),55(40), 11957.

[0006] Kahalalides: bioactive peptides from a marine mollusk Elysiarufescens and its algal diet Bryopsis sp. Hamann, Mark T.; Otto, CliftonS.; Scheuer, Paul J.; Dunbar, D. Chuck. Department of Chemistry,University of Hawaii of Manoa, Honolulu, Hi., USA J. Org. Chem. (1996),61(19), 6594-6600.

[0007]  [Erratum to document cited in CA125:190997]. J. Org. Chem.(1998), 63(14), 4856.

[0008] The marine environment: A resource for prototype antimalarialagents. El Sayed, Khalid A.; Dunbar, D. Charles; Goins, D. Keith;Cordova, Cindy R.; Perry, Tony L; Wesson, Keena J.; Sanders, Sharon C.;Janus, Scott A.; Hamann, Mark T. Center the Development NaturalProducts, University Mississippi, University, Miss., USA. J. Nat Toxins(1996), 5(2), 261-285.

[0009] The antitumoral compound Kahalalide F acts on cell lysosomes.Garcia-Rocha, Mar; Bonay, Pedro; Avila, Jesus. 28049-Madrid, Spain.Cancer Lett. (Shannon, Irel.) (1996), 99(1), 43-50.

[0010] Kahalalide F: a bioactive depsipeptide from the sacoglossanmollusk Elysia rufescens and the green alga Bryopsis sp. Hamann, MarkT.; Scheuer, Paul J. Dep. Chem., Univ. Hawaii, Honolulu, Hi., USA J. Am.Chem. Soc. (1993), 115(13), 5825-6.

SUMMARY OF THE INVENTION

[0011] We provide new formulations and new uses of kahalalide F.

PREFERRED EMBODIMENTS

[0012] A combination of a non-ionic surfactant and an organic acid issuited for use with a bulking agent to give a lyophilised form ofkahalalide F suited for reconstitution. Reconstitution is preferablyeffected with a mix of emulsifying solubiliser, alkanol and water.

[0013] The lyophilised composition preferably comprises mainly thebulking agent, such as at least go % or at least 95% bulking agentExamples of bulking agents are well known and include sucrose andmannitol. Other bulking agents can be employed.

[0014] The non-ionic surfactant in the lyophilised composition ispreferably a sorbitan ester, more preferably a polyethylene sorbitanester, such as a polyoxyethylene sorbitan alkanoate, especially apolyoxyethylene sorbitan mono-oleate, for example polysorbate 80. Thenon-ionic surfactant typically comprises a few % of the composition,such as 0 to 5% of the composition, for instance 2 to 3% of thecomposition.

[0015] The organic acid in the lyophilised composition is typically analiphatic acid, preferably a hydroxycarboxylic acid and more preferablya hydroxypolyearboxylic acid, notably citric acid. The organic acidtypically comprises a few % of the composition, such as 0 to 5% of thecomposition, for instance 2 to 3% of the composition.

[0016] The amount of kahalalide F in the lyophlised composition istypically less than 1%, or often less than 0.1%, of the mix. A suitableamount is in the range 50 to 200 μg, say about 100 μg, per 100 mg ofcomposition.

[0017] The emulsifying solubiliser for the reconstituting agent suitablycomprises an polyethylene glycol ester, notably an ester of a fattyacid, more preferably a PEG oleate such as PEG-35 oleate. Theemulsifying solubiliser is suitably 0 to 10% of the reconstitutingagent, typically about 3 to 7%, say about 5%. The alkanol is usuallyethanol, and is suitably 0 to 10% of the reconstituting agent, typicallyabout 3 to 7%, say about 5%. The remainder of the reconstituting agentis water, and gives a reconstituted solution suited for intravenousinjection.

[0018] Further dilution of the reconstituted solution with 0.9% salinemay be appropriate for infusion of the kahalalide F.

[0019] In a particularly preferred embodiment, the lyophilisedcomposition comprises 100 μg kahalalide F; 75 to 125 mg, especially 100mg, bulking agent; 1 to 3 mg, especially about 2 mg of acid; and 1 to 3mg, especially about 2 mg, of non-ionic surfactant.

[0020] The preferred reconstituting agent then comprises 2 to 7%, sayabout 5%, emulsifying solubiliser; 2 to 7%, say about 5%, alcohol; andremainder water.

[0021] The invention additionally provides kits comprising separatecontainers containing the lyophilised composition and the reconstitutingagent. Methods of reconstitution are also provided.

[0022] The present invention further provides a method of treating anymammal, notably a human, affected by cancer which comprisesadministering to the affected individual a therapeutically effectiveamount of a pharmaceutical composition thereof prepared byreconstitution of a lyophilised composition of this invention. Thepresent invention can be employed particularly for treatment of patientswith refractory cancers that do not respond favourably to othertreatments. In particular, the compositions of this invention can beemployed after other chemotherapy has been tried and not worked.

[0023] In one embodiment, the reconstituted solution is prepared forinfusion and is administered in a 3-hour infusion on concentrations ofup to around 20 or 25 μg/ml, typically up to 15 μg/ml. Suitable infusionequipment preferably includes a glass container, rather than one ofpolyethylene. Tubing is preferably of silicone.

[0024] A further aspect of this invention resides in new uses forkahalalide F. In particular, we envisage its use against prostate cancerand in particular androgen-independent prostate cancer, breast cancer,colon cancer, non-small cell lung cancer, ovarian cancer and fortreating neuroblastoma. Kahalalide F is also active againstdedifferentitated and mesenchymal chodrosarcomas and osteosarcomas. Thenew reconstituted formulations provided by this invention maybe employedfor the new uses, though other compositions are possible. Examples ofpharmaceutical compositions include any solid (tablets, pills, capsules,granules, etc.) or liquid (solutions, suspensions or emulsions) withsuitable composition or oral, topical or parenteral administration, andthey may contain the pure compound or in combination with any carrier orother pharmacologically active compounds. These compositions may need tobe sterile when administered parenterally.

[0025] Administration of the compounds or compositions of the presentinvention may be by any suitable method, such as intravenous infusion,oral preparations, intraperitoneal and intravenous administration. Weprefer that infusion times of up to 24 hours are used, more preferably2-12 hours, with 2-6 hours most preferred. Short infusion times whichallow treatment to be carried out without an overnight stay in hospitalare especially desirable. However, infusion may be 12 to 24 hours oreven longer if required. Infusion may be carried out at suitableintervals of say 2 to 4 weeks. In an alternative dosing protocol, thekahalalide F is administered for say about 1 hour for 5 consecuvitvedays every 3 weeks. Other protocols can be devised as variations.

[0026] Pharmaceutical compositions containing compounds of the inventionmay be delivered by liposome or nanosphere encapsulation, in sustainedrelease formulations or by other standard delivery means.

[0027] The correct dosage of the compounds will vary according to theparticular formulation, the mode of application, and the particularsitus, host and tumour being treated. Other factors like age, bodyweight, sex, diet, time of administration, rate of excretion, conditionof the host, drug combinations, reaction sensitivities and severity ofthe disease shall be taken into account. Administration can be carriedout continuously or periodically within the maximum tolerated dose.

[0028] The compounds and compositions of this invention may be used withother drugs to provide a combination therapy. The other drugs may formpart of the same composition, or be provided as a separate compositionfor administration at the same time or a different time. The identity ofthe other drug is not particularly limited, and suitable candidatesinclude:

[0029] a) drugs with antimitotic effects, especially those which targetcytoskeletal elements, including microtubule modulators such as taxanedrugs (such as taxol, paclitaxel, taxotere, docetaxel), podophylotoxinsor yinca alkaloids (vincristine, vinblastine);

[0030] b) antimetabolite drugs such as 5-fluorouracil, cytarabine,gemcitabine, purine analogues such as pentostatin, methotrexate);

[0031] c) alkylating agents such as nitrogen mustards (such ascyclophosphamide or ifosphamide);

[0032] d) drugs which target DNA such as the antracycline drugsadriamycin, doxorubicin, pharmorubicin or epirubicin;

[0033] e) drugs which target topoisomerases such as etoposide;

[0034] f) hormones and hormone agonists or antagonists such asestrogens, antiestrogens (tamoxifen and related compounds) andandrogens, flutamide, leuprorelin, goserelin, cyprotrone or octreotide;

[0035] g) drugs which target signal transduction in tumour cellsincluding antibody derivatives such as herceptin;

[0036] h) alkylating drugs such as platinum drugs (cis-platin,carbonplatin, oxaliplatin, paraplatin) or nitrosoureas;

[0037] i) drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors;

[0038] j) gene therapy and antisense agents;

[0039] k) antibody therapeutics;

[0040] l) other bioactive compounds of marine origin, notably thedidemnins such as aplidine or ecteinascidins such as Et 743.

EXAMPLES OF THE INVENTION

[0041] Experimental work which underlies the present invention isdescribed in the following Examples.

Example 1

[0042] Development of a Lyophilized, Parenteral PharmaceuticalFormulation of Kahalalide F, KF

[0043] The purpose of this study was to develop a stable parenteralformulation of KF to be used in early clinical studies.

[0044] Methods. Solubility and stability of KF were studied as afunction of polysorbate 80 (P80; 0.1-0.5% w/v) and citric acidmonohydrate (CA; 5-15 mM) concentrations using an experimental designapproach. Stabilities of KF lyophilized products containing crystalline(mannitol) or amorphous (sucrose) bulking agents were studied at +5° C.and +30° C. in the dark. Lyophilized products were characterized byinfrared (IR) spectroscopy and differential scanning calorimetry.Recovery studies after reconstitution of KP lyophilized product andfurther dilution in infusion fluid were carried out to select an optimalreconstitution vehicle.

[0045] Results. It was found that a combination of P80 and CA isnecessary to solubilize KF. Lyophilizcd products were considerably lessstable with increasing P80 and CA concentrations, with the P80concentration being the major effector. A combination of 0.1% w/v P80and 5 mM CA was selected for further investigation. Lyophilized productscontaining sucrose as bulking agent were more stable compared to theproducts containing mannitol. The glass transition temperature of thesucrose-based product was determined to be +46° C. The amorphous stateof the product was confirmed by IR analysis. A solution composed ofCremophor EL ethanol and Water for Injection (5/5/90% v/v/v CEW) kept KFin solution after reconstitution and further dilution with 0.9% NaCl(normal saline) down to 0.5 μg/mL.

[0046] Conclusions. A stable lyophilized formulation is presentedcontaining 100 μg of kahalalide F, 100 mg sucrose, 2.1 mg CA and 2 mgP80 to be reconstituted with a vehicle composed of 5/5/90% v/v/v CEW,and to be further dilute using normal saline.

Example 2

[0047] Compatibility and Stability of Kahalalide F in Infusion Devices

[0048] Kahalalide F is pharmaceutically formulated as lyophilizedproducts containing 50-150 μg active substance per dosage unit Prior toin i.v. administration it is reconstituted with a solution composed ofCremophor EL, ethanol absolute and Water for Injection (CEW, 5/5/90%v/w) with further dilution in 0.9% w/v sodium chloride for infusion. Theaim of this study was to investigate the compatibility and stability ofkahalalide F with different infusion systems prior to the start ofclinical trials. Due to the presence of Cremophor EL in the infusionsolution, leaching of diethylhexlphthalate from polyvinyl chlorideinfusion containers (PVC, Add-a-Flex®) was found. Loss of kahalalide Fas a consequence of sorption to contact surfaces was shown with aninfusion container composed of low density polyethylene (LD-PE,Miniflac®). We conclude that kahalalide F must be administered in a3-hour infusion in concentrations of 0.5 μg/mL to 14.7 μg/mL using anadministration set consisting of a glass container and silicone tubing.Kahalahde F 150 μg vial powder for infusion reconstituted with 5/5/90%v/v/v CEW is stable in the original container for at least 24 hours atroom temperature (+20-25° C.) and ambient light conditions. Infusionsolutions stored in glass infusion containers at either room temperature(+20-25° C., in the dark) or refrigerated conditions (+2-8° C., in thedark) are stable for at least 5 days after preparation.

Example 3

[0049] In Vitro Safety Toxicology of Kahalalide F

[0050] We have assessed the potential for toxicity of Kahalalide F, acompound derived from the Hawaiian mollusk, Ellysia rubefescens shown tohave potent chemotoxic effects against prostate and neu˜1 (Hcr2overexpressing) breast tumor cells.

[0051] Using the CellTiter96 (MTS, Promega) in vitro cytoxicity assay,Kahalalide exhibits little toxicity against cardiac (H9 c2 (2-1)) orskeletal muscle (L8) cells (LD₅₀=5 mM, 0.6 mM, respectively). Incontrast, Kahalalide-is cytotoxic to liver (AML-12), and kidney(NRX-52E) cells (LD₅₀=0.17 μM, 1.6 μM, respectively), and shows anintermediate toxicity to myelogenous stem cells (FDC-P1, LD₅₀=14 μM.These data are in close agreement with in vivo toxicity data. We havealso found this drug to be neuxotoxic at high concentrations in ourassay system and which correlates well with animal data showingneurotoxicity above the maximum tolerated dose (MTD). Using afluorescent viability stain (ethidium homodimer and calccin AM,Molecular Probes) coupled with immunocytochemistry, we determined that˜10 μM Kahalalide is toxic to central nervous system (CNS) neurons(neurofilament positive) but spares astrocytes (glial fibrilary acidicprotein positive) as well as sensory (substance P expressing) and motor(choline acetyl transferase positive) neurons in the spinal cord.

[0052] We conclude that Kahalalide F is a promising drug for thetreatment of prostate cancer as its neurotoxicity is relatively mild ator below MTD levels. Further, preliminary data indicate that KahalalideF may prove to be an ideal drug for treating neuroblastoma, ifdeliverable, due to its CNS neuron selectivity.

Example 4

[0053] Selective Antitumor Activity of Kahalalide F

[0054] Kahalalide F is a lysosomal poison with in vitro selectivity forhormone-independent prostate tumors, neu⁺ (Her2 overexpressing) breasttumor cells and neuroblastomas. An extended MoA includes inhibition oferbB2 and blocking of the EGF receptor as well as inhibition of TGF agene expression. Preclinical in vivo models have confirmed selectivityand sensitivity of hormone independent prostate tumors (PC-3 and DU-145)with a rodent MTD of 300 μg/kg BW. In vitro antiproliferative studiesshow equivalent IC₅₀ activities among certain prostate tumors (0.27 μMPC-3; 0.25 μM DU-145; 0.73 μM T-10, 0.24 μM DHM and 0.19 μM RB), but noactivity to hormone-sensitive LnCAP. Other studies show selective, butslightly less potent IC₅₀ activities to neu+breast tumor cellc (2.5 μMSK-BR-3: 2 μM BT-474) and to a neuroblastoma cell line (1 μM BE(2)C). Invitro exposure studies demonstrate that KF is not schedule-dependent Aminimum exposure of 1 hour is as potent as 48 hours, in most cases.Moreover, the immediate and delayed effects of cytotoxicity have thesame pharmacodynamics and do not increase with treatment duration. Phase1 trials incorporating a daily times five, weekly schedule will beginsoon in the evaluation of KF as a potential chemotherapeutic agentagainst solid tumors.

Example 5

[0055] Investigation of the Effects of Kahalalide F (PM92102) AgainstHuman Tumor Specimens Taken Directly from Patients.

[0056] In vitro studies have shown activity of KF to cause cell swellingand ultimately death (Garcia-Rocha, et al., Can Letters 99:43-50). Inthe present study, fresh human tumor specimens were treated with KF todetermine activity, utilizing the Human Tumor Cloning Assay. One hundredand four patient tumor specimens were treated with KF under 14-daycontinuous exposure at 0.01, 0.1. and 1.0 μM. Specimens were incubatedin a 2-layer soft-agar cloning system at 37° C. and were removed on Day14 for colony count. Colonies formed in the treated plates were comparedto the number of colonies formed in the untreated control plates, andthe percent colonies surviving at each concentration was calculated. Thepositive control plates contained the cell poison orthosodium vanadate(200 μg/ml). Among these specimens, approximately 30% were evaluablewith appropriate negative and positive controls. In vitro responses(inhibitory response indicated by 50% survival) were observed in 16%({fraction (5/31)}). 19% ({fraction (6/31)}), and 81% ({fraction(25/31)}) of the specimens at 0.01, 0.1, and 1.0 μg/M, respectively.There was a positive relationship between concentration and response toKF, with a significant response (81%) at the higher concentration tested(1.0>0.1>0.01). Notable responses were seen in breast (100%), colon(75%), non-small cell lung (100%), and ovarian cancer specimens (91%).KF is a promising anticancer agent, to which a broad spectrum of tumorsresponded.

Example 6

[0057] Phase I and Pharmacokinetic Study of Kahalalide F in PatientsWith Advanced Androgen Refractory Prostate Cancer

[0058] KF displays both in vitro and in vivo anti-tumor activity invarious solid tumor models including breast, colon, non-small cell lung,and in particular prostate cancer. On the basis of its selectivity, KFis now further developed as a potential anticancer agent againstandrogen independent prostate tumors.

[0059] OBJECTIVE: In the present phase I clinical and pharmacokinetic(PK) study the toxicity profiles PK and anti-tumor activity of KF areinvestigated.

[0060] METHODS: KF is administered as an intravenous infusion over onehour, during five consecutive days every three weeks in patients withadvanced or metastatic androgen refractory prostate cancer. On the basisof the MTD values defined in mice, a starting dose of 20 μg/m2/day wasselected, which is equivalent to a total dose of 100 μg/m2. PK of KFwere determined in plasma during the first course. Bioanalysis of KF wasperformed by LC-MS/MS. LDH, AF and especially PSA levels of each patientwere also evaluated during the study to determine the activity of KF.

[0061] RESULTS AND DISCUSSION: At present 7 patients have beenregistered. Patients had a median age of 66 years (range 54-75). Onepatient per level was entered at 20, 40, 80 and 160 μg/m2/day. Due totransaminase elevation a number of 4 patients were entered at thecurrent dose level, 320 μg/m2/day. The first patient of this study wasre-entered at this dose level. Observed adverse events were rapidlyreversible mild headache, fatigue, pain and local edema. The only drugrelated toxicity to date was a rapidly reversible CTC grade 3 ASAT thatoccurred at 320 μg/m2/day. PK revealed a linear relationship betweendose and AUC over the whole dose range. Total plasma clearance was 267mL/min (±115) and the terminal half-life of intravenous KF in thesepatients was 0.46 h (±0.13). Maximum plasma concentrations reached atthe current dose level (35-50 ng/mL) are potentially active for prostatecancer in the clonogenic tumor assay (activity from 15 ng/mL). Thus far,the schedule is well tolerated. One patient showed a significantdecrease in PSA level (>50%) associated to clinical improvement (painrelief). Two additional patients experienced minor PSA reductions, onestill ongoing after two cycles. The maximum tolerated dose has not beenreached yet and the study is ongoing.

Example 7

[0062] Kahalilide F is cytotxic to dedifferentitated and mesenchymalchodrosarcomas, CHSAs, and osteosarcomas, OSAs, as well as tohepatocellular and prostate carcinoma cells. It did not significantlyinhibit the growth of CHSA and OSA cells, and was able to elicit acytotoxic effect even if exposed to cells for as short as 10 minutes.

1. A formulation of kahalalide F comprising a lyophilised mix ofkahalide F, a non-ionic surfactant, an organic acid and a bulking agent.2. A kit comprising a formulation of claim 1, together with instructionsfor dilution with a reconstitution solution of a mix of emulsifyingsolubiliser, alkanol and water.
 3. The kit of claim 2, together with asupply of the reconstitution solution.
 4. A reconstituted solutionprepared from a formulation of claim 1 or a lit of claim 2 or
 3. 5. Areconstituted solution according to claim 4, when reconstituted using areconstitution solution of a mix of emulsifying solubiliser, alkanol andwater.
 6. A diluted reconstituted solution comprising a reconstitutedsolution according to claim 4 or 5 when diluted with 0.9% saline to aconcentration suited for infusion of the kahalalide F.
 7. The use of adiluted reconstituted solution according to claim 6, in the treatment ofcancer.
 8. The use of kahalalide F against prostate cancer, breastcancer, colon cancer, non-small cell lung cancer, ovarian cancer, fortreating neuroblastoma, or against dedifferentitated or mesenchymalchondrosarcomas or osteosarcomas.
 9. The use according to claim 7 or 8,wherein the kahalalide F is administered by infusion about 1 hour for 5consecutive days every 3 weeks.
 10. The use according to claim 7, 8 or9, wherein the kahalide F is adminstered in combination with anotherdrug.
 11. The use according to any of claims 7 to 10, wherein thekahalalide F is administered to a refractory patient.